DESCRIPTION (Investigator's Abstract): Covalent lipid modifications anchor numerous signaling proteins, including heterotrimeric G proteins, nonreceptor tyrosine kinases and Ras to the cytoplasmic face of the plasma membrane where they convey signals from cell surface receptors to intracellular effectors. The posttranslational attachment of lipids to these signal transducers is necessary for their function. Palmitoylation (S-acylation) is the only covalent lipid modification that is reversible and is a regulated modification. The broad goal of this project is to understand the biology and enzymology of protein palmitoylation in signal transduction using biochemical and genetic approaches. Heterotrimeric G proteins have been used as a model to understand the functional significance of palmitoylation. In the previous grant period, a protein palmitoytransferase activity that acylates G-protein alpha subunits and Ras was identified and characterized. In this application, purification of this enzyme and cloning of its cDNA is proposed. A genetic analysis of G-protein palmitoylation in yeast revealed that both alpha and gamma subunits are modified with palmitate and provided evidence for the importance of palmitoylation in G-protein function. These studies established the basis for the genetic screens proposed in this application to identify genes that are required for palmitoylation of G-protein subunits, a necessary first step in characterizing the physiological role of G-protein palmitoyltransferases. To increase our understanding of the organization of signaling proteins in cellular membranes, the hypothesis that palmitoylation of G proteins is responsible for their targeting to specialized regions of the plasma membrane will be explored. The association of modified and unmodified G proteins with membranes enriched in glycolipids and cholesterol will be studied in artificial membranes and in cells. The recently discovered family of proteins called Regulators of G protein Signaling, or RGS proteins, revealed a novel mode of regulating signal transduction. We determined that RGS4 is palmitoylated in a domain of the protein that is required for its plasma membrane localization. The studies proposed in this application will investigate the mechanism of membrane association of RGS4 and the function and regulation of RGS4 palmitoylation.